CN114746436A

CN114746436A - FGF2 polypeptide with improved temperature stability and protease resistance and application thereof

Info

Publication number: CN114746436A
Application number: CN202080081543.4A
Authority: CN
Inventors: 李廷贤; 任亨淳; 安泳俊; 李卿源; 郑叡恩; 车基元; 李元揆; 禹珠娘
Original assignee: Korea Institute of Ocean Science and Technology KIOST
Current assignee: Korea Institute of Ocean Science and Technology KIOST
Priority date: 2019-11-25
Filing date: 2020-11-16
Publication date: 2022-07-12
Anticipated expiration: 2040-11-16
Also published as: EP4067374A1; CN114746436B; JP2023505655A; US20230076299A1; WO2021107473A1; EP4067374A4; US12103953B2; JP7441948B2

Abstract

The present invention provides a polypeptide having FGF2 activity and improved temperature stability and protease resistance. The polypeptide comprises at least one substitution selected from the group consisting of substitution of aspartic acid (D) at positions 1 to 28 of the sequence with glutamic acid (E), substitution of cysteine (C) at position 78 with isoleucine (I) or leucine (L), and substitution of cysteine (C) at position 96 with isoleucine (I) or tryptophan (W).

Description

FGF2 polypeptide with improved temperature stability and protease resistance and application thereof

Technical Field

The present disclosure relates to FGF2 polypeptides with improved temperature stability and protease resistance and uses thereof.

Background

FGF (fibroblast growth factor) is a factor that plays an important role in regulating cell growth, proliferation, and differentiation. In order to maintain the functions of various tissues of the human body, various types of FGFs are produced, which exert unique functions in cell differentiation and proliferation. However, with age, the concentration of FGF in various tissues such as the skin gradually decreases, and thus the regeneration and division functions of cells are weakened, resulting in wrinkles in the skin and decreased elasticity.

Among the various FGFs, FGF2(Fibroblast Growth Factor 2) is composed mainly of 155 amino acids and has a molecular weight of approximately 18 kDa. FGF2 has a broad mitogenic (mitogenic) and cell survival activity (cell survival activity) and plays a powerful mediator in wound healing, angiogenesis and nervous system growth.

Therefore, FGF2 has been developed not only as a drug for promoting angiogenesis, wound healing, cartilage formation or bone formation, neurogenesis (neurogenesis), but also as a cosmetic raw material for skin regeneration, wrinkle removal, or elasticity increase. In addition, since it has a function of maintaining cells in a pluripotent state, it is added as a main factor to a medium for culturing human Pluripotent Stem Cells (PSC).

FGF2, which has various functions in humans as described above, is reported to be less thermodynamically stable than epithelial cell growth factor (EGF), insulin-like growth factor (IGF), and Vascular Endothelial Growth Factor (VEGF). In addition, there is a problem that the protease is easily cleaved. Therefore, in order to make FGF2 suitable for industrial use, ensuring thermodynamic stability and/or protease resistance of FGF2 is an indispensable condition.

Disclosure of Invention

Technical problem

The present disclosure is directed to providing FGF2 polypeptides with improved temperature stability and protease resistance.

The present disclosure is directed to a pharmaceutical or cosmetic composition comprising an FGF2 polypeptide having improved temperature stability and protease resistance.

The present disclosure is directed to a human pluripotent stem cell culture medium containing an FGF2 polypeptide having improved temperature stability and protease resistance.

Technical scheme

The FGF2 polypeptide with improved temperature stability according to the embodiments is a temperature stability improving (thermal stable) polypeptide comprising at least one substitution selected from the group consisting of substitution of aspartic acid (D) from position 1 to position 28 of the sequence with glutamic acid (E), substitution of cysteine (C) at position 78 with leucine (L) or isoleucine (I), substitution of cysteine (C) at position 96 with tryptophan (W) or isoleucine (I), and having activity inherent to FGF 2.

The composition according to the embodiments comprises a polypeptide that improves temperature stability and protease resistance and a pharmaceutically or cosmetically acceptable carrier.

The human pluripotent stem cell culture medium according to the example contains a polypeptide that improves temperature stability and protease resistance as an active ingredient.

Effects of the invention

The FGF2 polypeptides of the examples, when produced as products, exhibit improved temperature stability and protease resistance when compared to wild-type human FGF2 polypeptides.

Unlike the existing wild-type human FGF2 product, polypeptides that improve temperature stability and protease resistance also maintain activity during circulation and storage. Therefore, it can be used as an active ingredient of a pharmaceutical or cosmetic composition. In addition, when used as an effective ingredient of a culture medium for human pluripotent stem cells, it has an advantage that the activity of inducing undifferentiated proliferation can be maintained for a long period of time, as compared with wild-type FGF 2.

Drawings

FIG. 1 shows the polypeptide of wild-type FGF2 (SEQ ID NO: 1).

FIG. 2 shows SDS-PAGE of wild-type FGF2 and FGF2 variants (pQE80_ hFGF2(S137P), pQE80_ hFGF2(D28E, S137P)).

Fig. 3 shows SDS-PAGE for testing stability at 37 ℃ of wild-type FGF2(Δ 9N-hFGF2) and FGF2 variants (Δ 9N-hFGF2(D28E, S137P),. Δ 9N-hFGF2(D28E, C78L, C96I, S137P),. Δ 9N-hFGF2(D28E, C78I, C96I, S137P),. Δ 9N-hFGF2(D28E, C78L, C96W, S137P),. Δ 9N-hFGF2(D28E, C78I, C96W, S137P)).

Fig. 4 shows SDS-PAGE for testing stability at 45 ℃ of wild-type FGF2(Δ 9N-hFGF2) and FGF2 variants (Δ 9N-hFGF2(D28E, S137P),. DELTA.9N-hFGF 2(D28E, C78L, C96I, S137P),. DELTA.9N-hFGF 2(D28E, C78I, C96I, S137P),. DELTA.9N-hFGF 2(D28E, C78L, C96W, S137P),. DELTA.9N-hFGF 2(D28E, C78I, C96W, S137P)).

FIG. 5 is a bar graph measuring the change in cell proliferation activity at 37 ℃ for wild-type FGF2 and FGF2 variants (pQE80_ hFGF2(S137P), pQE80_ hFGF2(D28E, S137P)).

FIG. 6 is a bar graph measuring changes in cell proliferation activity at 37 ℃ of wild-type FGF2 (. DELTA.9N-hFGF 2) and FGF2 variants (. DELTA.9N-hFGF 2(D28E, S137P),. DELTA.9N-hFGF 2(D28E, C78L, C96I, S137P),. DELTA.9N-hFGF 2(D28E, C78I, C96I, S137P).

FIG. 7 is a bar graph measuring changes in cell proliferation activity at 42 ℃ of wild-type FGF2 (. DELTA.9N-hFGF 2) and FGF2 variants (. DELTA.9N-hFGF 2(D28E, S137P),. DELTA.9N-hFGF 2(D28E, C78L, C96I, S137P),. DELTA.9N-hFGF 2(D28E, C78I, C96I, S137P).

FIG. 8 shows SDS-PAGE for detecting resistance to protease of wild-type FGF2 (. DELTA.9N-hFGF 2).

FIG. 9 shows SDS-PAGE for detecting protease resistance of FGF2 variants (. DELTA.9 _ hFGF2D28E + C78L + C96I + S137P).

Detailed Description

Hereinafter, the embodiments will be described in detail so that those skilled in the art can easily practice the present invention. The embodiments can be implemented in numerous different ways and are not limited to the specific embodiments described herein.

Unless certain terms used in the present disclosure are defined otherwise below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The methods and processes described in this disclosure are generally performed according to conventional methods provided herein. Generally, nomenclature used in molecular biology, biochemistry, analytical chemistry, and cell culture, and laboratory procedures are well known in the art and are the same as commonly used.

Variants

The present disclosure provides FGF2 polypeptides that are thermostable via site-directed mutagenesis. In the present disclosure, mutants are prepared by site-directed mutagenesis after rational prediction of the optimal amino acid at unknown new positions by bioinformatic analysis and in silico protein design.

Figure 1 shows the wild-type human FGF2 polypeptide sequence.

In the present disclosure, the term "wild-type" refers to native FGF2 having the amino acid sequence most prevalent among members of a species. In the present disclosure, wild-type FGF2 is human FGF2, which is an 18kDa protein of 155 amino acids in length (position 1 of the sequence, fig. 1).

In the present disclosure, "fragment" refers to a functional fragment of FGF2 polypeptide having FGF2 activity. Furthermore, a "fragment" refers to a functional fragment of an FGF2 polypeptide having a sequence identity of 85% or more with the sequence at position 1 of the sequence. Fragments of FGF2 polypeptides further comprise at least one substitution according to the invention. Preferably, the sequence identity is at least 96%, 97%, 98%, 99% or 100%. By fragment is meant a polypeptide consisting only of the complete polypeptide sequence and partial structure, with possible C-terminal or N-terminal deletions of the variant. Such functional fragments may have a cell binding region of the target FGF2 protein according to the invention and a heparin-binding fragment.

In the present disclosure, "sequence identity" means that identical amino acid residues are found in the FGF2 polypeptide according to the invention as described above. The wild-type human FGF2 polypeptide is used as a reference when aligned to a specific amino acid sequence corresponding to a reference molecule after alignment of specified contiguous fragments of the amino acid sequence of FGF2 polypeptide. The percentage of sequence identity was calculated as follows: the percentage of sequence identity is calculated by detecting the number of positions at which the identical amino acid residue is present in both sequences, calculating the number of positions that are identical, dividing by the total number of positions of the fragment aligned to the reference molecule, and multiplying by 100. Methods of sequence alignment are well known in the art. The reference sequence as used in the present application refers to the particular corresponding wild-type human FGF2 protein according to the present invention. For example, FGF2 is highly conserved for mammalian species such as mouse, rat, rabbit, primate, pig, dog, cow, horse, and human, and exhibits more than 85% sequence identity across a wide range of species. Preferably, the sequence identity is at least 96%, 97%, 98% or 99% or more or 100%. It will be appreciated by those skilled in the art that less than 15% of the remaining amino acids in the full length of the FGF2 protein according to the invention are variable, for example, due to the use of a different source of FGF2 species or the addition of suitable non-FGF 2 peptide sequences or tags or the like commonly known in the art. FGF2 proteins according to embodiments of the present invention that have over 85% identity relative to wild-type FGF2 have other members of the FGF family that typically have very low sequence identity and therefore are unlikely to contain other proteins than similar FGF 2.

The present inventors confirmed that the 28 th site or the 137 th site in wild-type human FGF2 is a position related to thermostability and/or protease resistance of FGF2 polypeptide, and that the 78 th site or the 96 th site of cysteine exposed to the surface of FGF2 is a position related to thermostability and/or protease resistance.

The replacement of the most suitable amino acid at the position associated with thermostability and/or protease resistance requires the inventors' efforts (innovative step).

The present inventors confirmed that thermal stability can be improved by substituting glutamic acid (E) for aspartic acid (D) at position 28.

The present inventors also confirmed that thermal stability can be further improved by substituting serine (S) at position 137 with proline (P).

The present inventors have also confirmed that thermal stability can be further improved by simultaneously substituting the 28 th site and the 137 th site.

The stability after 19 mutations was compared by SDM ((http:// mark. bioc.cam. ac. uk, University of Cambridge) and Discovery studio 2019(BIOVIA) at positions 78 and 96, and confirmed thereby.

In the result of SDM analysis, when the mutation was leucine (L), the Predicted value (Predicted pseudo. DELTA.G) was 0.33 and the Predicted value was the highest, and when the mutation was isoleucine (I) in Discovery studio, the mutation energy change value (kcal/mol) was-1.20 and the mutation was expected to be the most stable.

In the result of SDM analysis, the Predicted value (Predicted pseudo. DELTA.G) was 0.19 and the Predicted value was the highest when the mutation was isoleucine (L) at position 96, and the mutation energy change value (kcal/mol) was-0.39 and was expected to be the most stable when the mutation was tryptophan (W) in Discovery studio.

US9169309, US2017-0291931, EP3380508, US20180319857 and the like disclose that cysteine at position 78 is substituted with serine (S), tyrosine (Y), or cysteine at position 96 is substituted with serine (S), tyrosine (Y), threonine (T), asparagine (N), or the like. Most of these substituted amino acids are hydrophilic and uncharged amino acids, while the substituted amino acids in the present disclosure are hydrophobic amino acids, which are considered to fall within the category not easily anticipated by the prior art.

Thus, in the present disclosure, the achievable variant may be any one of the various variants shown in table 1 below.

[ TABLE 1 ]

In each of the variants described above, although mutations at a single position may also improve thermostability and/or protease resistance, more than 2 mutations may be more beneficial in improving thermostability and/or protease resistance. Further, 3 to 4 mutations may be more advantageous in improving thermostability and/or protease resistance. Generally, the gene encoding FGF2 is cloned and then expressed in a transformed organism, preferably a microorganism. The host organism expresses the foreign gene under expression conditions to produce FGF 2. In addition, synthetic recombinant FGF2 can be formed in eukaryotes such as yeast and human cells. FGF2 may be in the form of 146 amino acids, 153-155 amino acids, or a mixture thereof, depending on the recombinant production method. The present application demonstrates for the first time that partial changes in wild-type FGF2 will create FGF2 mutations with greater temperature stability and long half-life than the wild-type protein. The FGF2 protein according to the invention for inserting the substitutions described in the present application can be derived from any mammal, such as mouse, rat, rabbit, primate, pig, dog, cow, horse and human, provided that the criteria specified in the present application are met (i.e. having the preferred biological activity of wild-type FGF2 and being thermostable). Preferably, the target FGF2 protein is derived from human origin. However, all biologically active variants of mammalian FGF2 having a sequence identity of greater than 85%, most preferably greater than about 96%, greater than 97%, greater than 98%, or greater than 99%, relative to the amino acid sequence of the human FGF2 protein at position 1 of the sequence used as a standard of comparison, are useful in the present invention.

In some embodiments, a stabilized FGF2 polypeptide described herein in accordance with the present invention can further comprise a sequence or tag other than any other FGF peptide known in the art for use in detecting, purifying, labeling a particular tissue or cell, improving stability, prolonging activity, improving expression, and the like.

Pharmaceutical and cosmetic compositions

The various variants disclosed in table 1 may be provided as pharmaceutical and/or cosmetic compositions with a pharmaceutically or cosmetically acceptable carrier.

The various variants disclosed in table 1 may be administered to a subject in need of promoting angiogenesis, promoting wound healing, promoting chondrogenesis or bone formation, or promoting neurogenesis, or a subject in need of improving skin conditions such as improving wrinkles, improving skin elasticity, preventing skin aging, preventing hair loss or promoting hair growth, improving skin moisture, removing age spots, or treating acne. The various variants disclosed in table 1 may be administered in the "raw state" or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, etc., but the salts, esters, amides, prodrugs or derivatives may be selected from pharmacologically suitable substances, i.e. substances effective for the present method. Salts, esters, amides, prodrugs and other derivatives of the peptides are known to those skilled in the art of synthetic organic chemistry and may be prepared, for example, by standard procedures known.

The various variants disclosed in table 1 may be formulated in transdermal application type products such as aerosols, creams (creams), serums (serums), and patches for subcutaneous, parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or topical administration. The compositions may be administered in a variety of unit dosage forms depending on the method of administration. Suitable unit dosage forms can include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained release formulations, lipid complexes, and the like.

When the various variants disclosed in table 1 are combined with a cosmetically acceptable carrier to form a cosmetic composition, fillers such as hyaluronic acid fillers, Polymethylmethacrylate (PMMA) microspheres, and collagen fillers, and the like may be further included. The present compositions may preferably be administered topically, subcutaneously or transdermally.

The present composition may be an injectable composition.

The composition may further comprise collagen (e.g., bovine, porcine, human), hyaluronic acid. The collagen may be synthetic collagen and the hyaluronic acid may be cockscomb or a fermentation product of a microorganism.

The present compositions may also include an anesthetic (e.g., lidocaine).

The present compositions may be skin creams (e.g., face creams).

The present composition may be a liquid preparation in the form of whey or a toner.

The present compositions may be gelatinous, semi-solid preparations.

A pharmaceutically acceptable carrier may comprise a carrier for animals, more particularly humans or animals, more particularly humans, which is approved by a federal or state regulatory agency, or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia. By "carrier" is meant a diluent, adjuvant, excipient, adjuvant or vehicle (vehicle), e.g., administered with one or more peptides described herein.

The pharmaceutically acceptable carrier may, for example, contain more than one physiologically acceptable compound which acts to stabilize the composition or to increase or decrease absorption of the various variants disclosed in table 1. Physiologically acceptable compounds may for example comprise carbohydrates (such as glucose, sucrose or dextran), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, protection and absorption enhancers (such as lipids), compounds or other excipients which reduce the clearance or hydrolysis of peptides, stabilizers and/or pH adjusting buffers.

In particular, other physiologically acceptable compounds used in the manufacture of tablets, capsules, gel caps, and the like may include, but are not limited to, binding agents, diluents/fillers, disintegrants, lubricants, and suspending agents.

Excipients, optional disintegrants, binders, optional lubricants, and the like can be added to the various variants disclosed in table 1 and the resulting composition compressed to produce an oral dosage form (e.g., a tablet). If desired, the compressed product can be coated using known methods for taste blocking or enteric or sustained release.

Other physiologically acceptable compounds that can be formulated into dosage forms with the various variants disclosed in table 1 can include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. The excipient can be sterilized and used without contamination.

The various variants disclosed in table 1 may be incorporated into cosmetic dosage forms and topically applied, and may be formulated as skin creams (e.g., face creams) or lotions, wrinkle reduction creams, or may be incorporated into cosmetics, sun screens, or moisturizers.

Additionally, the various variants disclosed in table 1 can be incorporated into dosage forms that optionally further comprise fillers, humectants, vitamins (e.g., vitamin E), and/or colorants/dyes.

Suitable injectable cosmetic dosage forms may include, but are not limited to, dosage forms incorporating the various variants disclosed in table 1 along with one or more filler materials. Exemplary materials that can be used as injectable cosmetic wrinkle fillers can include, but are not limited to, temporary (absorbent) fillers such as collagen (e.g., synthetic collagen, bovine collagen, porcine collagen, human collagen, etc.), hyaluronic acid gel, calcium hydroxide apatite (typically implanted in gel form), or poly-L-lactic acid (PLLA), among others. The peptides may also be incorporated into injectable cosmetic formulations containing permanent (non-absorbable) fillers. Illustrative "permanent" fillers may include, but are not limited to, polymethylmethacrylate beads (PMMA microspheres).

The various variants disclosed in table 1 may be incorporated into or administered with dermal fillers, injectable dosage forms: such injectable dosage forms may further comprise an anesthetic (e.g., lidocaine or an analog thereof). The injectable dosage forms are substantially sterile or aseptic or conform to guidelines of the subcutaneous injection filler administration.

The various variants disclosed in table 1 can be administered to a subject using any route known in the art, for example, including injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, or intradermal), inhalation, transdermal administration, rectal administration, vaginal administration, or oral administration. Preferred routes of administration include subcutaneous, transdermal or topical administration.

An effective amount of each of the variants disclosed in table 1 can be administered by local (i.e., non-systemic) administration such as via peripheral administration including, but not limited to, peripheral intramuscular, intralinear, and subcutaneous administration.

Administration of the various variants disclosed in table 1 may be by any convenient means, such as injection, intravenous and arterial stents (including eluting stents), catheter, oral, inhalation, transdermal administration, rectal administration, and the like.

For each of the variants disclosed in table 1, prior to administration, a dosage form may be formulated with a pharmaceutically acceptable carrier, as described above. For pharmaceutically acceptable carriers, one part is determined by the particular composition to be administered and the particular method used to administer the composition.

In the context of the methods described herein, the dose administered to a subject should be sufficient to produce a beneficial therapeutic response (e.g., increased subcutaneous adipogenesis) in the subject over time. The dosage depends on the efficacy of the particular vehicle/delivery method employed, the site of administration, the route of administration and the condition of the subject, as well as the body weight or surface area of the subject to be treated. The size of the dose also depends on the presence, nature and extent of any adverse side effects associated with the administration of a particular peptide in a particular subject.

The various variants disclosed in table 1 may be administered systemically (e.g., orally or as an injection) according to standard methods well known to those skilled in the art. The peptides can be applied to the oral cavity in various forms such as lozenges, aerosols, mouthwashes, coated swabs, and the like. Various oral and sublingual dosage forms are also contemplated. When the various variants disclosed in table 1 are formulated as injections, they may be administered in a long acting (depot) dosage form to provide treatment over a period of time.

The various variants disclosed in table 1 may be applied, for example, topically to a skin surface, a local lesion or wound, a surgical site, and the like.

Conventional transdermal drug delivery systems, i.e., transdermal "patches," may be used to deliver the various variations disclosed in table 1 through the skin, and may be contained within a laminate structure typically provided as a drug delivery device that is applied to the skin.

Other dosage forms for topical delivery include, but are not limited to, ointments, gels, sprays, liquids, and creams. Ointments may be semisolid preparations, which are usually based on petrolatum or other petroleum derivatives. As with other carriers or vehicles, the ointment base should be inert, stable, non-irritating, and non-sensitizing. The creams containing selected variants disclosed in Table 1 may generally be viscous liquid or semisolid emulsions, often oil-in-water or water-in-oil. Cream bases are typically water-washable and contain an oil phase, an emulsifier, and an aqueous phase. As will be appreciated by those skilled in the art, the particular ointment or cream base to be used is the one provided for optimal drug delivery.

For the various variants disclosed in table 1, it may be provided as a "concentrate" in a storage container ready for dilution (e.g., in a pre-measured volume) or as a "concentrate" in a soluble capsule ready to be added to a large volume of water, alcohol, hydrogen peroxide, or other diluent. For example, the peptide may be lyophilized for subsequent reconstitution.

The various variants disclosed in table 1 may have a variety of uses. The various variants disclosed in table 1 may have utility in a wide variety of applications. For example, subcutaneous fat provides skin with fullness and firmness and thus has utility in cosmetic surgery to enhance subcutaneous adipogenesis. Aged skin contains less subcutaneous fat. Thus, one or more of the various variants disclosed in table 1 described herein are applied to the desired site to promote the formation of subcutaneous fat, making the skin plumper and younger appearing. This method can replace existing methods of transplanting adipocytes from other parts of the human body (e.g., thighs or buttocks) which generally have a low success rate.

The various variants disclosed in table 1 can be administered to selectively improve subcutaneous adipose tissue (e.g., for improving subcutaneous adipose tissue without substantially increasing visceral fat and/or other adipose tissue). In response to administration of the various variants disclosed in table 1, adipocytes are formed in dermal fibroblasts, and volume can be increased within a selected subcutaneous site in a subject.

The various variants disclosed in table 1 can be used to reduce scarring. This may be accomplished by applying one or more of the variants disclosed in table 1 above in an amount sufficient to reduce the scar site and/or improve the appearance of the scar site. The scar may be, for example, a scar resulting from a burn, a scar resulting from surgery, a scar resulting from acne, a scar resulting from biopsy, or a scar resulting from injury.

The various variants disclosed in table 1 may for example be used in various cosmetic processes to improve the appearance of skin. This may be accomplished by applying one or more peptides to the site of the subject in an amount sufficient to improve the appearance of the skin. Such administration may comprise subcutaneous administration to an area such as the lips, eyelids, cheeks, forehead, chin, neck, etc. The peptides may be used in methods of reducing wrinkles, reducing sagging skin, improving the surface texture of skin, reducing, removing or filling wrinkles, removing or reducing age spots, and/or removing fundus dark circles. These cosmetic applications are exemplary and not limiting.

The various variants disclosed in table 1 may be used to improve tissue volume at a site in a subject. This may be achieved by administering one or more of the peptides described herein in an amount sufficient to increase the tissue volume at the site of the subject. For example, increasing the tissue volume may include compacting or augmenting breast tissue and/or compacting or augmenting hip tissue or other parts of the body or face.

In this case, FGF2 may be used in an amount of 0.01 to 10 ppm. If it exceeds 10ppm, an excessive amount may cause side effects. Therefore, the practical range is 0.01 to 10ppm, preferably 0.01 to 2 ppm.

The various variants disclosed in table 1 may also be used to soften the skin within a subject area. This may be accomplished by applying one or more of the peptides described herein in an amount sufficient to soften the skin at the desired site. The softening may comprise softening the skin of acne causing scars, softening cellulite areas, softening or reducing stretch marks, and/or smoothing wrinkles.

The various variants disclosed in table 1 may be used to mobilize stem cells to form subcutaneous fat in a subject. This can be achieved by administering the variants disclosed in table 1 in an amount sufficient to mobilize stem cells to form subcutaneous fat. This has utility, for example, in various reconstructive surgical procedures.

The various variants disclosed in table 1 may be used to reconstruct tissue in a subject. Such reconstructions may include, for example, breast reconstructions (e.g., after surgery to remove a tumor), or facial or limb reconstructions (e.g., after an automobile accident or burn). This can be achieved by applying the various variants disclosed in table 1 in an amount sufficient to increase the tissue volume during or after tissue reconstruction. The various variants disclosed in table 1 may optionally be used with tissue graft materials or other methods of improving skin or injured tissue treatment.

For the various variants disclosed in table 1, can be used to reduce heel pain in a subject by administering an amount sufficient to reduce the heel pain experienced by the subject while walking.

Various variants disclosed in table 1 may be administered to increase subcutaneous fat for increasing thermoregulation and/or improving immune function. Various variants disclosed in table 1 may be treated to prevent disease or to treat progressive diseases associated with increased organ fat, including but not limited to cardiovascular disease and other diseases associated with obesity.

Any of these methods may be topical or systemic and may be performed by any of the routes described herein, e.g., topical, subcutaneous, transdermal, oral, nasal, vaginal, and/or rectal administration. Preferably, the variants disclosed in table 1 may be administered by subcutaneous injection. Alternatively, the various variants disclosed in said table 1 may be administered topically in the form of a skin cream, such as a cream, or transdermally via a transdermal patch.

Although the above uses and methods have been described in connection with the use of the human body, they are also applicable to animals, such as veterinary uses. Thus, preferred organisms include, but are not limited to, humans, non-human primates, canines, equines, felines, porcines, ungulates, rabbits, and the like.

Culture medium

The content of each variant disclosed in table 1 may be a "medium effective amount" to provide the human pluripotent stem cell medium, which corresponds to the amount required to maintain the pluripotent stem cells in an undifferentiated state during at least 5 subcultures.

In the present disclosure, the term "human pluripotent stem cell" encompasses human embryonic stem cells and derived pluripotent stem cells, characterized by self-renewal capacity (self-renewal capacity), the capacity to form the same self-offspring and pluripotency of almost all cell types that allow the generation of the human body.

In the present disclosure, the term "maintaining stem cells in a pluripotent state" refers to maintaining cells in an undifferentiated state having the ability to differentiate into almost all cell types. This pluripotent state depends on a mixture (cocktail) of the most important growth factor, FGF2, which is a growth factor supporting desiccation. FGF2 supports self-regeneration by several means: the mitogen-activated protein kinase pathway is directly activated and indirectly promotes transforming growth factor β 1 and activin signaling (Greber et al, 2008, Stem Cells 25, 455-464). FGF2 promotes pluripotency of human PSCs in a variety of ways through cell attachment and survival functions (Eisellova et al, 2009, Stem Cells 27, 1847-.

The present disclosure provides for the characterization of target FGF2 suitable for engineering, demonstration of substitution effects in proteins, methods of utilizing proteins in human PSC cultures, and media containing one or more thermostable FGF2 proteins described in this application for culturing human PSCs in an undifferentiated state. Human Embryonic Stem Cells (ESCs) employed in the examples provided in this application were derived from blastocyst-stage embryos obtained with informed consent from donors. A well-characterized human ESC Cell Line (Adewumi et al, 2007, Nat Biotechnol 25, 803-816) was used at 29-41 passages, CCTL14(Centre of Cell Therapy Line). For example, human Induced Pluripotent Stem Cells (iPSCs), the AM13 cell strain derived by reprogramming of skin fibroblasts transfected with Yamanaka's cocktail and Sendai virus (Sendai virus transfection) based on mountain mixtures was used in a state of 34-41 passages (Kruta et al, 2014, Stem Cells and Development 23, 2443-2454).

Hereinafter, preferred experimental examples will be given to aid understanding of the present invention, but the following experimental examples are only illustrative of the present invention and the scope of the present invention is not limited to the following examples.

Experimental example 1: construction, purification and thermostability analysis of mutants Using pQE80 vector

Mutations at 1 position (S137P) and 2 positions (D28E, S137P) of FGF2 were synthesized and subcloned into pQE80L vector with His-Tag. The recombinant vector into which FGF2 was inserted was transformed into osetta (DE3) pLysS cells for expression.

Was inoculated into 10ml of LB medium (Ambrothia) (0.25 g was used), and 10ul of Ampicillin (Ampicillin, 50mg/ml) was added, followed by preculture at 37 ℃.

10ml of the preculture broth and 1ml of Ampicillin (Ampicillin, 50mg/ml) were inoculated into 1L of LB medium (ambothia) (25 g was used), and main culture was carried out at 37 ℃. When the OD600 value was 0.6, the culture was cooled in a refrigerator at 4 ℃ for 10 minutes, and then 0.5mM of beta-D-1-thiogalactopyranoside (IPTG) was added, thereby obtaining E.coli cells that were induced to express at 20 ℃ for 20 hours.

The expressed pQE80_ FGF2 was dissolved in lysis buffer (20mM Tris pH 8.0,200mM NaCl,3mM DTT), sonicated, centrifuged at 13000 r.p.m. for 30 minutes, and then purified.

After centrifugation, the final solubilized supernatant was injected into a column with heparin beads. The first purification was performed by elution with 60ml of elution buffer (elution buffer,20mM Tris pH 8.0,1800mM NaCl,3mM DTT) using 3 times the volume of pQE80_ FGF2 protein injected into the column, in a first wash (wash) buffer (20mM Tris pH 8.0,200mM NaCl,3mM DTT) and a second wash (wash) buffer (20mM Tris pH 8.0,500mM NaCl,3mM DTT).

Finally, the pQE80_ FGF2 protein fragment was purified by gel filtration using HiLoadTM 16/60 Superdex 75(Amersham Biosciences) column and 1XPBS buffer (137mM NaCl,2.7mM KCl,10mM Na2HPO4,2mM KH2PO4, pH 7.4), (WELGENE).

The purified FGF2 protein was reacted at 37 ℃ for 0,2, 4, and 6 days using substantially 1XPBS buffer at a concentration of 0.5mg/ml, stained with Coomassie brilliant blue staining reagent, and subjected to 15% SDS-PAGE, and the results are shown in FIG. 2.

As shown in fig. 2, it was found from the FGF2 polypeptide band confirmed by 15% SDS-PAGE that stability on SDS-PAGE was improved for both mutant polypeptides (pQE80_ hFGF2(S137P), pQE80_ hFGF2(D28E, S137P)) compared to hFGF2 (wild-type) polypeptide without mutation.

Experimental example 2: construction, purification and thermostability analysis of mutants Using pET17b vector

Mutations at 1 position (S137P), 2 positions (D28E, S137P), and 4 positions ((D28E, C78L, C96I, S137P), (D28E, C78L, C96W, S137P), (D28E, C78I, C96I, S137P), (D28E, C78I, C96W, S137P)) of FGF2 were synthesized and subcloned into pET17b vector with His. The recombinant vector with FGF2 inserted was transformed into Rosetta (DE3) pLysS cells for expression.

The expressed pET17b _ FGF2 was dissolved in a lysis buffer (20mM Tris pH 8.0,200mM NaCl,3mM DTT), subjected to sonication, centrifuged at 13000 r.p.m. for 30 minutes, and then purified.

After centrifugation, the final solubilized supernatant was injected into a column with heparin beads. The first purification was performed by elution with 60ml of elution buffer (elution buffer,20mM Tris pH 8.0,1800mM NaCl,3mM DTT) using 3 times the volume of pET17b _ FGF2 protein injected into the column, in a first wash (wash) buffer (20mM Tris pH 8.0,200mM NaCl,3mM DTT) and a second wash (wash) buffer (20mM Tris pH 8.0,500mM NaCl,3mM DTT).

For the 4 position mutants, purification was performed in affinity chromatography using a solution formulated in buffer and elution buffer without 3mM DTT.

Finally, pET17b _ FGF2 protein fractions were purified by gel filtration using HiLoadTM 16/60 Superdex 75(Amersham Biosciences) column and 1XPBS buffer (137mM NaCl,2.7mM KCl,10mM Na2HPO4,2mM KH2PO4, pH 7.4), (WELGENE).

Stability test at 37 deg.C

The purified FGF2 protein was reacted at 37 ℃ for 0, 3, 6, and 9 days using substantially 1XPBS buffer at a concentration of 0.5mg/ml, stained with Coomassie brilliant blue staining reagent, and subjected to 15% SDS-PAGE, and the results are shown in FIG. 3.

As shown in FIG. 3, the thermostability of the variants was improved as seen from the band of FGF2 polypeptide confirmed by 15% SDS-PAGE.

For density detection, the density of the SDS-PAGE gel was measured using the imageJ program (Wanyne Rasband), the results of which are shown in Table 2 below.

[ TABLE 2 ]

Variants	Day 0	3 days	6 days	9 days
					△9N_hFGF2	100	84	69	65
△9N_hFGF2(D28E,S137P)	100	100	65	17
					△9N_hFGF2(D28E,C78L,C96I,S137P)	100	86	102	89
△9N_hFGF2(D28E,C78I,C96I,S137P)	100	98	102	103
					△9N_hFGF2(D28E,C78L,C96W,S137P)	100	99	96	94
△9N_hFGF2(D28E,C78I,C96W,S137P)	100	112	92	65

In%, it can be seen from the results in table 2 that the thermostability of the 4-position variant was improved compared to wild-type hFGF 2. In particular, Δ 9N-hFGF2(D28E, C78L, C96I, S137P), Δ 9N-hFGF2(D28E, C78L, C96W, S137P), Δ 9N-hFGF2(D28E, C78I, C96I, S137P) are relatively more thermostable than the other variants.

Stability test at 45 deg.C

For the purified FGF2 protein, it was reacted at 45 ℃ for 0,1, 2, 3, 4, 5, 6 days using substantially 1XPBS buffer at a concentration of 0.5mg/ml, stained with Coomassie blue staining reagent, and subjected to 15% SDS-PAGE, the results of which are shown in FIG. 4.

As shown in FIG. 4, the thermostability of the variants was improved as seen from the band of FGF2 polypeptide confirmed by 15% SDS-PAGE.

For density detection, the density of the SDS-PAGE gel was detected using imageJ program (Wanyne Rasband), the results of which are shown in table 3 below.

[ TABLE 3 ]

From the results in table 3, it is clear that the thermostability of the variants was improved compared to wild-type hFGF 2. In particular, Δ 9N-hFGF2(D28E, C78L, C96I, S137P), Δ 9N-hFGF2(D28E, C78I, C96I, S137P) are relatively more thermostable than the other variants.

Experimental example 3: confirmation of cell proliferation Capacity of mutant Using pQE80 vector

BALB3T3 cells were used for the mutants prepared by the same method as in Experimental example 1, and cultured and maintained in DMEM medium containing 10% bovine serum. To confirm the cell proliferation activity based on FGF2, cells were cultured in F12/medium containing 10ug/ml insulin (insulin), 1uM dexamethasone (dexamehasone), 10ug/ml transferrin (transferrin), 10ng/ml sodium selenite (sodium selenite), 100ug/ml ovalbumin (ovalbumin), 5ug/ml fibronectin (fibronectin).

The number of cultured cells in a 96-well plate was 0.5X10⁴Perwell and treated with heparin (10ug/ml) and FGF2(0.3ng/ml) for 42 hours. For the increase in cell number, WST-8[2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt was used]The extent of production of WST-8 formazan (formazan) by electron mediator (electron mediator) and intracellular dehydrogenases (dehydrogenases) was detected and confirmed. The degree of production of WST-8 formazan was confirmed by absorbance (450 nm). The experiment was repeated 3 times and expressed as "mean + standard deviation". After the FGF2 protein was stored at 37 ℃ for 0,2, 4, and 6 days, changes in cell proliferation activity were confirmed.

The results are illustrated in fig. 5. Referring to FIG. 5, a decrease in activity was observed after hFGF2 was stored at 37 ℃ for 2 days, and less than 60% of the activity of the protein was observed after storage at 37 ℃ for 4 days at 37 ℃ for 0 days. On the other hand, the activity of the hFGF 2S 137P variant was observed to be lower than that of hFGF2 after 2 days of storage at 37 ℃ and 60% of the protein was observed to have less activity than that of 0 day of storage at 37 ℃ after 4 days of storage at 37 ℃. On the other hand, the higher the date of storage at 37 ℃ the hFGF2D28E + S137P variant had lower activity, but the activity reduction was much less than that of hFGF2 and hFGF 2S 137P variants.

Experimental example 4: confirmation of cell proliferation Capacity of the mutant Using pET17b vector

The mutant prepared by the same method as in experimental example 2 was used BALB3T3 cells, and cultured and maintained in DMEM medium containing 10% bovine serum. To confirm the cell proliferation activity based on FGF2, cells were cultured in F12/medium containing 10ug/ml insulin (insulin), 1uM dexamethasone (dexamehasone), 10ug/ml transferrin (transferrin), 10ng/ml sodium selenite (sodium selenite), 100ug/ml ovalbumin (ovalbumin), 5ug/ml fibronectin (fibronectin).

The number of cultured cells in a 96-well plate was 0.5X10⁴Perwell and treated with heparin (10ug/ml) and FGF2(0.3ng/ml) for 42 hours. For the increase in cell number, WST-8[2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt was used]The extent of production of WST-8 formazan (formazan) by electron mediator (electron mediator) and intracellular dehydrogenases (dehydrogenases) was detected and confirmed. The degree of production of WST-8 formazan was confirmed by absorbance (450 nm). The experiment was repeated 3 times and expressed as "mean + standard deviation". After FGF2 protein was stored at 37 ℃ for 0, 3, 6, 9, and 12 days, changes in cell proliferation activity were confirmed.

The results are illustrated in fig. 6. Referring to FIG. 6, Δ 9_ hFGF2 (wild type) showed a decrease in activity after 3 days of storage at 37 ℃ and was confirmed to have almost lost activity after 6 days of storage at 37 ℃. The Δ 9_ hFGF2D28E + S137P variant had longer activity than the wild-type protein and decreased activity after 9 days of storage at 37 ℃. In contrast to this, neither the Δ 9_ hFGF2D28E + C78L + C96I + S137P mutein nor the Δ 9_ hFGF2D28E + C78I + C96I + S137P variant had an activity decrease observed when stored at 37 ℃ for 12 days. It can be confirmed that the variant according to the example stably maintained the activity of the protein at 37 ℃.

After the FGF2 protein was stored at 45 ℃ for 0,1, 2, 3, 4, 5, 6, and 7 days, respectively, changes in cell proliferation activity were confirmed. The results are illustrated in fig. 7. Referring to FIG. 7, Δ 9_ hFGF2 (wild type) showed a sharp decrease in activity after 1 day of storage at 45 ℃ and almost lost activity after 2 days of storage at 45 ℃. The Δ 9_ hFGF2D28E + S137P mutein had a longer activity than the wild-type protein, but the activity decreased after 2 days of storage at 45 ℃. In contrast to these, the activities of Δ 9_ hFGF2D28E + C78L + C96I + S137P mutein and Δ 9_ hFGF2D28E + C78I + C96I + S137P mutein gradually decreased according to the storage date at 45 ℃, but maintained longer than the wild type, and maintained the proliferative activity ability more than 2 times even after 6 days of storage at 45 ℃.

Experimental example 5: confirmation of resistance to protease cleavage of mutants utilizing pET17b vector

Δ 9_ hFGF2 (wild type) and Δ 9_ hFGF2D28E + C78L + C96I + S137P muteins prepared by the same method as in Experimental example 2 were examined for resistance to protease cleavage.

FIGS. 8 and 9 show the results of SDS-PAGE detecting Δ 9_ hFGF2 (wild type) and Δ 9_ hFGF2D28E + C78L + C96I + S137P muteins, respectively. In FIGS. 8 and 9, No.1 is the result of detection without incubation after protease treatment, No.2 is the result of detection after incubation at 37 ℃ for 3 hours after protease treatment, and Nos. 3 to 14 are the results of detection after incubation at 37 ℃ for 3 hours after treatment with 0.0025mg/mL of 12 different proteases for 0.25mg/mL of FGF2, respectively.

For the density detection of each band, the density of the SDS-PAGE gel was detected using imageJ program (Wanyne Rasband), and the results are shown in Table 4 below.

[ TABLE 4 ]

From the results of fig. 8 and 9 and the contents of table 4, it is clear that the Δ 9_ hFGF2D28E + C78L + C96I + S137P mutant protein has an improved resistance to protease as a whole as compared with Δ 9_ hFGF2 (wild type). In particular, the resistance to alpha-chymotrypsin (alpha-C), Trypsin (TR), Subtilisin (SU), proteinase K (P-K), clostripain-Arg-C (CL), Thermolysin (TH), actin (A-E) is relatively increased.

In the foregoing, various embodiments have been described, but the scope of the claims is not limited to the embodiments. Various modifications may be made to the specific embodiments within the scope of the present description and the accompanying drawings, which naturally fall within the scope of the claims.

Industrial applicability

Can be applied to the technical field of cosmetics or medicines.

<110> KIOST

KBIO HEALTH

<120> FGF2 polypeptide having improved temperature stability and protease resistance and use thereof

<130> OPP20203831KR

<150> KR 10-2019-0152362

<151> 2019-11-25

<150> KR 10-2020-0129526

<151> 2020-10-07

<160> 1

<170> KoPatentIn 3.0

<210> 1

<211> 155

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly

1 5 10 15

Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu

20 25 30

Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg

35 40 45

Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu

50 55 60

Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn

65 70 75 80

Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys

85 90 95

Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr

100 105 110

Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys

115 120 125

Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys

130 135 140

Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser

145 150 155

Claims

1. A polypeptide having improved temperature stability, wherein,

the polypeptide comprises at least one substitution selected from the group consisting of substitution of aspartic acid (D) from position 1 to position 28 of the sequence with glutamic acid (E), substitution of cysteine (C) at position 78 with isoleucine (I) or leucine (L), and substitution of cysteine (C) at position 96 with isoleucine (I) or tryptophan (W), and has FGF2 activity.

2. The polypeptide for improving temperature stability according to claim 1,

serine (S) at position 137 is substituted with proline (P).

3. The polypeptide for improving temperature stability according to claim 2, wherein,

the substitutions are 2, 3 or 4.

4. The polypeptide for improving temperature stability according to claim 3, wherein,

aspartic acid (D) at position 28 is substituted with glutamic acid (E),

the cysteine (C) at position 78 is substituted with isoleucine (I) or leucine (L),

the cysteine (C) at position 96 is substituted with isoleucine (I),

the serine (S) at position 137 is substituted with proline (P).

5. The polypeptide for improving temperature stability according to claim 3, wherein,

aspartic acid (D) at position 28 is substituted with glutamic acid (E),

the cysteine (C) at position 78 is substituted with an isoleucine (I),

the cysteine (C) at position 96 is substituted with isoleucine (I),

the serine (S) at position 137 is substituted with proline (P).

6. The polypeptide for improving temperature stability according to claim 1,

the polypeptide has at least 85% or more sequence identity to position 1 of the sequence or a fragment thereof.

7. A composition, comprising:

a polypeptide according to any one of claims 1 to 6 which improves temperature stability; and

a pharmaceutically or cosmetically acceptable carrier.

8. A culture medium for human pluripotent stem cells, comprising the polypeptide having improved temperature stability according to any one of claims 1 to 6 as an active ingredient.